Lee A L, Wand A J
Johnson Research Foundation, Department of Biochemistry & Biophysics, University of Pennsylvania, Philadelphia 19104, USA.
J Biomol NMR. 1999 Feb;13(2):101-12. doi: 10.1023/a:1008304220445.
The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin-spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of -170 +/- 15 ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1 ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6 ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived tau m values for proteins. The near identity of observed T2 and T1 rho values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine tau m values. It is also noted that the reliable determination of tau m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as tau m increases.
研究了影响通过核磁共振弛豫方法可靠且高效地测定描述蛋白质分子重排相关时间的各种因素。已在17.6、14.1、11.7和9.4特斯拉下测定了泛素中15N NMR弛豫的核Overhauser效应、自旋晶格和自旋 - 自旋弛豫参数。这组异常宽泛的弛豫参数使得能够研究化学位移各向异性、无模型光谱密度的函数形式以及所测定的自旋 - 自旋弛豫参数的可靠性对蛋白质整体翻滚特征描述的影响。将15N化学位移各向异性（CSA）视为可调参数，当使用无模型光谱密度拟合来自所有场的T1和NOE数据时，发现化学位移张量宽度的共识值为 -170±15 ppm，全局各向同性相关时间为4.1 ns。在全局相关时间的测定中纳入T2弛豫参数会使其增加到4.6 ns。这种明显的不一致可能解释了蛋白质的NMR和荧光衍生的τm值之间经常发现的大部分差异。观察到的T2和T1ρ值几乎相同，这表明慢运动的贡献不是与所获得的T1和NOE数据明显不一致的根源。各种考虑表明，这种明显差异的根源可能在于除了与这些弛豫参数测量相关的常见实验困难之外，零频率下光谱密度的贡献未被简单的无模型形式主义所表示。最后，仅使用T1和NOE数据获得了泛素的轴对称扩散张量。就可用于可靠测定τm值的弛豫数据类型和组合达成了一项建议。还指出，随着τm增加，从15N T1和NOE弛豫参数可靠测定τm值将变得越来越困难。
